Lab Internship: Week 4

Tuesday 7/26
I just discovered that I was not supposed to culture cells, according to the safety advisor for the building.  So technically, I was not doing something that I was supposed to do.  Victor will now have to prepare all of the cells needed for experimentation.  Victor is planning on doing an experiment testing how TNF (tumor necrosis factor) affects cell behavior.  TNF is a cytokine that regulates many cell functions, such as proliferation, apoptosis, coagulation, etc.  In this experiment, Victor explained that the TNF will excite the cells and may cause them to jump around and possibly out of the traps.  I prepared the cells in four devices with Hoechst stain so we can identify the cells and I introduced TNF into two of the four devices, yet the trapping efficiency was not 100% in all devices.  Victor said we will have to image specific regions of the channel to see how the TNF affected the cells.  We just need to see the cells, so the trapping efficiency is irrelevant.

Wednesday 7/27
Victor asked me to accompany him to the stock room to get some supplies.  I assumed the stock room was in the basement of the building, but it was really in KBT.  The stock room was reminiscent of a corner store, like a drug dealer's grocery store.  Walter White would probably love the stock room.  There are two on Yale's primary campus: one at KBT and one in the med school.  There is also one at West Campus, but that's far away (at like West Haven or something).

There was also a lab meeting where one of the labmates, Linda Fong, presented her progress on her project on how p38, a mitogen-activated protein kinase, affects HIV latency and apoptosis of HIV infected immune cells, which could lead to more effective forms of therapy.

I also began to learn ImageJ to analyze the images of the devices I took with Victor.  I need to learn how to use ImageJ and develop a macro to count the amount of cells in the image and differentiate between the cells.  CellProfiler is another program I can use, but I do not know how to use it.  I asked Andrés for help, but he was busy with his project.

Victor and I were about to image the devices with cells for the TNF experiment, but we found out that the RPMI evaporated in the incubator and the cells died as a result.  Many traps in some of the devices also disappeared.  Victor guessed that this occurred because there was not enough humidity in the incubator.  Tomorrow, we will begin repeating the experiment by preparing the devices.

I found a way to analyze an image and count the cells.  I just need to use the Color Threshold function to "select" the cells and analyze the particles with the Analyze Particles function.  I then get a table full of data with the measurements of each particle.  I already tested this method with images from the efficiency experiment.  The method was accurate.  I will use this method with my experiment to count the cells and further test the efficacy of this method.

Thursday 7/28
I prepared four flow-patterning devices for Laura and her experiment.  I cut them out into individual devices, then I cleaned them.  I also punched holes in the inlets and outlets (20 inlets, 20 outlets for each device).

I prepared four more devices for my experiment (take 2).  I used J65C 6.6 for my experiment, not 4.4 since 6.6 was thinner and less clumpy and dense.  I prepared two of the four devices as controls, and the other two as the manipulated group.  I will stimulate the cells in the manipulated group with TNF to see how TNF affects cell behavior.  According to Victor, the TNF will excite the cells in a way that will make the cells jump out of the traps.  I will leave the cells in the devices, and leave the devices in an incubator for 24 hours.  I will also put a humidifier (fancy term for water in a bowl) with the devices to ensure that the RPMI will not evaporate so the cells do not die.

Friday 7/29
Most of the cells from the devices all disappeared.  We do not know why.  It was perfectly fine when we incubated the device.  Maybe the humidifier loosened the bonding of the devices?  We checked the outlet and everything, but nothing was different--besides the fact the most of the cells were gone.  It's not like they planned a prison break or anything.  Cells can't do that--I think.  There were a few cells I could image in specific regions of the devices, so I imaged them.  The control devices were plain blue because of the Hoechst stain, but the TNF activated HIV in almost 100% of the cells, so that made for some pretty pictures.  It almost calmed me of the stress and shame of having almost another failed experiment.  Victor told me this happened all the time, which just made me feel bad for scientists everywhere.  Now, I have to understand my data and learn what it means.  It's likely that the TNF was an activator for transcription, allowing HIV to be transcribed and become active in the cell, but it could also be that TNF activated a specific MAPK (mitogen-activated protein kinase) pathway.  MAPK pathways are involved in many cellular activities, such as gene expression, proliferation, mitosis, apoptosis, etc.